The stability of tryptophan and tyrosine hydroxylases was studied in vitro. The activity of tryptophan hydroxylase decays rapidly when preincubated at 37 C for up to 60 min. The inclusion of tetrahydrobiopterin (BH4), 6-MPH4, or dimethyl-tetrahydropterin (DMPH4) in the preincubation mixture significantly protects tryptophan hydroxylase from 02-thermal inactivation. The protective effects were provided only by reduced cofactor since biopterin did not prevent inactivation. Tyrosine hydroxylase, on the other hand, is stable throughout a 60 min preincubation and the inclusion of BH4, 6MPH4, or DMPH4 in the preincubation causes a loss of catalytic activity. The inhibitory substance is not a peroxide or superoxide anion, nor is it the cofactor or catecholamine end-product. It appears that exposure of tyrosine hydroxylase to its cofactor BH4 in the absence of the substrate tyrosine leads to the loss of activity.